Auto focusing is an essential feature in many automated inspection fields such as the chip industry, biomedical research, data reading/recording in optical information carriers, etc.
Auto-focusing techniques used in the inspection/measurement of such structures as semiconductor wafers typically utilize an optical system separated from an imaging optics, which projects a light pattern (e.g., spots, lines, etc.) on the wafer and optimizes the image of this pattern (by using intensity contrast or displacement). Such techniques are applicable only for long working distance microscopes, i.e., with small numerical apertures. These techniques require calibrations and adjustments to match auto-focusing and imaging optical systems, and are hard to implement, especially when the imaging optics has variable magnification. U.S. Pat. No. 5,925,874 describes a solution to the variable magnification problem based on projecting a focusing pattern through a magnifying imaging optics.
Other common methods for auto-focusing in the chip industry inspection are based on the detection of light reflected from a specimen, wherein incident light is focused either on the outer surface of the specimen, or on inner interfaces of the specimen (as in the case of infrared light).
A recently developed auto-focusing technique for use with measurement/inspection on semiconductor wafers is disclosed in U.S. Pat. No. 6,172,349. This technique is useful in both conventional microscopy and interferometry. According to this technique, an operator designates the area within each field of view where the measurement has to be taken, and, for each area of interest (where the microscope is to be focused), translates the microscope along its optical axis (Z-axis) while measuring the image intensities at discrete sub-areas within the area of interest. These image intensities are then evaluated, and those having the greatest signal-to-noise ratio and occurring at a common point along the Z-axis are selected, and the corresponding sub-areas are identified. During subsequent inspections of the area of interest, only light reflected from the identified sub-areas is used to focus the microscope.
Numerous publications (e.g., Born M. and Wolf, E. Principles of optics Cambridge University Press, 1997; T. Wilson and C. Sheppard, “Theory and practice of scanning optical microscopy”, Academic Press, 1984) disclose optical principles, based on detecting the intensity variation above and below a focal plane (z-profile) for a point-like source, which are the basis for many auto-focusing systems.
U.S. Pat. No. 4,595,829 discloses a technique for use with reflected light microscopes. This technique utilizes the production of a measuring point on the surface of an object by an eccentric measuring beam formed by blocking a portion of the path of a full beam, and imaging the measuring point onto a photoelectric device by reflecting the measuring beam along the blocked-out path.
U.S. Pat. No. 4,844,617 discloses an auto-focusing technique for use in the inspection of semiconductor wafers. Here, a confocal microscope utilizes auto-focusing based on maximal intensity evaluated through an aperture larger than the diffraction limited size.
U.S. Pat. No. 5,288,987 describes a focusing technique for stereo microscopy, utilizing the imaging of an incident beam through a cylindrical lens projecting a thin bar-shaped marking on a viewed object surface. In this technique, imaging and focusing are not carried out via the same optics. confocal microscope on the most reflecting layer by an iterative z-scanning of the intensity profile.
U.S. Pat. No. 6,128,129 discloses an auto-focusing technique for a microscope based on the optical path difference to a front focused position and to a back position that are conjugate with respect to the image forming point of the image forming optical system.
U.S. Pat. No. 4,798,948 describes an auto-focusing arrangement for use with incident light microscopes of dark field illumination. According to this technique, an incident light beam is comprised of several (at least two) partial beams which coincide in at least a central region of the incident light beam and which are of different wavelengths. An auto-focus measurement beam is presented by one of these partial beams. A lens element is inserted into the central region of the incident beam so as to change the beam cross-section (converging and diverging).
Some auto-focusing techniques (e.g., U.S. Pat. Nos. 5,995,143 and 5,790,710; and Geusebroek et al., Cytometry, 39:1–9 (2000) and references cited therein) are based on the analysis of image contrast and local intensity distributions. These techniques typically require the acquisition and analysis of many images to reach the best focus. The application of these techniques for fluorescence microscopy of biological samples is described in U.S. Pat. No. 6,259,080, where the maximal signal to background value criterion is used. Here, a shutter is added to block the excitation illumination between image acquisition periods, thus minimizing the sample bleaching. The focusing technique of U.S. Pat. No. 5,790,710 is intended for use in a scanning microscope for inspecting fluorescently stained biological samples. This technique is aimed at avoiding bleach and phototoxicity utilizing the phase-contrast microscope image.
Auto-focusing techniques utilizing an increase of the depth of focus of a focusing optics by digital combination of series of images taken at many close focal planes are disclosed, for example, in the following publications: Haeusler, G. and Koemer, E., “Imaging with Expanded Depth of Focus”, Zeiss Inform., Oberkochen, 29:9–13, No.98E., (1986/87); Schormann, T. and Jovin, T. M., “Contrast Enhancement and depth perception in three-dimensional representation if differential interference contrast and confocal scanning laser microscope images.”, J. Microscopy 166:155–168, (1992). According to these techniques, a plurality of images is taken and processed three-dimensionally to find the sharpest features of all focal planes which are presented in the single “deep focus” image. Additionally, these techniques are not preserving intensities, and therefore are not applicable for quantitative fluorescence microscopy applications.
Present automated high-resolution (high-content) high-throughput applications of light microscopy in biology require scanning a large number of samples, such as cell yeast or bacteria, typically inside multi-well micro-plates, and acquiring images in each well which resolve intracellular details at the utmost resolution possible by the optics. In order to resolve such details, setting the focus within the vertical focal resolution of the objective is mandatory. This means 0.3–0.5 μm for immersed objectives, and 0.6–0.9 μm for high-numerical aperture long-working-distance air objectives. Such accuracy is far beyond the tolerance of the bottom surfaces of multi-well dishes, and even typical high-performance XYZ stages cannot scan distances of about 10 cm in the XY plane while keeping the Z coordinate within the above tolerances.
A practical solution for the existing instruments is to find the focus for a given sample, based on the optimization of image sharpness. Different algorithms, employing Fourier frequency space and real space image analysis, have been used, and Z scan, in a range that is expected to include the optimal focus, can guarantee that the best focal plane will be found (Geusebrock et al., Cytometry 39:1–9 (2000) and references 1–8,10–11,25–26 cited therein). To accelerate the process, the scan is first made in large steps, and refined till the required focus accuracy is reached. However, this approach has several inherent problems:
1. The process is slow. Even if processing is performed in real time, at least an order of magnitude slower throughput is expected as compared to maximum possible image acquisition rate.
2. A focus search algorithm could be based on efficient optimization. For example, Fibonacci minimization can reduce the range of the minimum search from 0.3 mm to 0.3 μm in 12 steps. Yet, there is no scheme that will map the distance from the focus into any evaluation parameter with smooth dependence displaying a single minimum over three orders of magnitude range of defocusing. Scans must therefore start close to the focus (typically within 20 μm) to refine its position
3. Sharpness criteria are very sensitive to dirt, scratches and fingerprints on surfaces. Glass slides or plastic well bottoms are a fraction of a millimeter thick and may often cause auto-focus devices based on maximal sharpness to focus at the wrong side of the sample substrate.
4. Sharpness algorithms are context-dependent, and there is no global focus criterion that can equally apply to a wide variety of image types. Fluorescently tagged cells may be found by the virtue of the cell-specific staining. However, image-based focusing is not applicable to fluorescent microscopy, where repeated imaging may cause bleach and phototoxicity. Fluorescence is localized also to specialized sub-volumes, thus the intensity varies greatly and is sample dependent. Typical biological screens based on fluorescent labels need to report intensities over a widely distributed range, as well as their absence. Focusing methods that evaluate fluorescence are therefore not applicable for such screens.
5. Biological specimens (such as live cells) are basically transparent, and have very low reflectivity, usually from structures (such as organelles) dispersed through their whole volume. Transmitted light microscopy can acquire images by optical contrasting methods (such as DIC or phase contrast) that avoid photo-toxic fluorescence excitation. Yet, even if the focus reaches a plane somewhere within the extent of the cell height, typically 4–10 μm, some sub-cellular details may still be out-of-focus. To assure the covering of the whole cell height by three-dimensional (3D) optical sectioning methods, one needs to over-scan a focal range about twice as high as the cell, since the initial plane may be close to the bottom or the top of the cell. Phase images may have the best contrast at focal heights that depend on irregular structures within or above the sample, and hence, cellular details may still be blurred for high resolution imaging. Phase images may also produce the best contrast at under- or over-focus conditions due to the nature of phase-optics and due to the misalignment of the phase ring optics.